The invention relates to the preparation of samples for viewing with an electron microscope and, more particularly, it relates to a microcentrifugation tube for the concentration of samples of cells for examination under an electron microscope.
A continual problem encountered by many investigators in in scientific research is the obtaining of adequate samples for experimentation and subsequent processing for electron microscopy. This is especially true for those investigators who work with cell cultures, bacteria cultures, and viral samples. It is a special problem where experimental samples are obtained in individual lots from human volunteers wherein the quantity of the sample is often limited and the obtaining of multiple samples is often difficult or impossible.
The adequacy of the sample size is critical because of the number of steps through which a sample must go during the preparation of the sample for examination by an electron microscope. In particular, the processing of a sample may include fifteen to twenty steps, each of which entails centrifuging the sample to separate and remove unwanted matter. Unfortunately, each of these steps may result in a significant loss of sample cell population to the point wherein the sample is essentially lost.
Specifically, the preparation of a sample may include the steps of first removing a sample of cells from the human subject and centrifuging the cells to collect them. A protein fixative is then added to cross-link the proteins, following which the sample is rinsed twice to remove any of the unreacted protein fixative. Of course, each of these rinses involves the use of the centrifuge to remove the rinse and the unreactive fixative. A fixative for the membranes is then added, following which the sample is again rinsed twice to remove any unreacted membrane fixative. The sample is then rinsed with ethanol for as many as seven different concentrations of ethanol in order to remove any water in the sample. A commercial imbedding medium is then added, which is typically a plastic, in two different concentrations, each of which are substantially removed from the sample, again by centrifugation. The sample is then mixed with a final imbedding medium and an accelerator and the sample and imbedding medium are centrifuged to pull the sample cells to the bottom of the mixture. The imbedding medium is then treated (usually by heating) to cause the medium to harden into a hard polymerized plastic which is the final sample product.
This final plastic product is then sliced on a microtome in sections having a thickness on the order of 500 to 900 .ANG. for viewing under an electron microscope.
It should be emphasized that because of the centrifugation of the sample following each of the steps the potential for loss of part of the sample cell population in each step is significant. Accordingly, if the beginning sample cell population is small, the sample may be essentially lost before the procedure is through. Indeed, it has been the experience of the present inventors that for some samples, the preparation of the sample so diminished the sample cell population that the resulting product was essentially worthless for meaningful examination.
A contributing cause to the loss of cell population during processing is the tube currently available and most commonly used to prepare the final sample. Specifically, the tube currently used has a bottom cross-sectional area which is too large to be suitable for the slicing of the sample on the microtome. From this lower cross-sectional area, the inner walls of the tube flair at an included angle of approximately 45.degree. for a given distance at which the walls proceed upwardly parallel to each other. It has been found that for the angle of approximately 45.degree., the sample cells tend to gather along the walls of the tube instead of migrating to the bottom of the tube as desired. Accordingly, the opportunity for the loss of sample cell population at each step is magnified by the number of cells which adhere to the gently sloping sides.
The migration of cells to the sloping side is disadvantageous for yet another reason. In particular, since the capsules presently available have a lower cross-sectional area which is too large for slicing in the microtome, the final sample product must be trimmed at the bottom in order to allow the insertion of the sample into the microtome for slicing. Accordingly, those cells which have migrated and adhered to the gently sloping sides will almost invariably be trimmed from the sample in the final preparation of the sample for slicing in the microtome.
The capsule presently available therefore enhances the loss of cells during each early preparatory step of the sample and almost invariably insures the loss of some cells during the trimming of the sample for slicing on the microtome.
It is therefore desirable to provide a tube which both minimizes the congregation of cells along its sides and prevents the loss of cells by trimming for slicing in the microtome.